Elucidation of tumor suppressor function provides much valuable insights into the mechanism of tumorigenesis. We have previously identified a novel phosphatase, TEP1, that shares substantial homology to several cytoskeletal proteins such as tensin. The gene encoding TEP1 is mapped to chromosome 10q23, a locus frequently deleted in human cancers. TEP1 is identical to the candidate tumor suppressor PTEN or MMAC1, which was isolated by positional cloning method. PTEN/MMAC1/TEP1 is deleted or mutated in many primary human cancers including glioblastma, endometric tumor, breast and prostate cancers and in several cancer predisposition syndromes such as Cowden disease. The broad, long term objective of this application is to elucidate the molecular mechanisms by which the cellular signaling pathways are linked to the regulatory processes for cell cycle progression and cell survival. In particular, this proposal is focused on investigating the mechanism by which the tumor suppressor protein PTEN functions to regulate cell cycle progression and how the inactivation of this tumor suppressor contributes to the genesis of human cancer. Our recent studies strongly suggest that in vivo, PTEN acts as a specific phosphatase towards phosphatidylinositol 3,4,5-trisphosphate (PIP3). We have generated the mouse embryonic stem cells in which the Pten gene was deleted by homologous recombination (Pten-/- cells). We have shown that Pten-/- cells contained elevated level of PIP3 and increased activation of Akt/PKB, a downstream signaling molecule in the PI 3-kinase pathway. Consequently, Pten-/- cells exhibited enhanced cell proliferation and cell survival. We further demonstrated that PTEN deletion caused advanced cell cycle progression into S-phase and a critical target for PTEN effect on cell cycle is p27KIP1, a CDK inhibitor. We propose to elucidate the molecular mechanism by which PTEN regulates p27 level and cell cycle progression. Our specific aims are: 1) To determine how the PTEN pathway regulates p27 level; 2) To analyze the PI 3-kinase downstream factors involved in regulation of p27; 3) To establish biochemical and molecular assays to identify the PTEN responsive factors that control p27; 4) To investigate the regulation of PTEN activity using structure and function analysis. The proposed research program in this application should provide novel insights into the molecular mechanisms that underlie the PTEN tumor suppressor function. Such knowledge should also help to provide a molecular basis for designing novel strategies for the diagnosis and treatment of human cancer.